It has long been known that the genetic origin and the physiological status of a cell can be determined and investigated by studying its genome, its transcriptome, its proteome and its methylome.
The term “genome” denotes the totality of the heritable nucleic acids in a sample, i.e. an organism, a tissue, a cell or a cell compartment or a biopsy, a smear, a section or the like. Generally the heritable nucleic acid is DNA.
The term “transcriptome” denotes the sum of the genes transcribed in such a sample, i.e. transcribed from DNA to RNA, at a given point of time, and thus the totality of all RNA molecules.
The term “proteome” denotes the totality of all proteins in said sample.
The term “methylome” describes the methylation profile of the genome. It comprises the totality and the pattern of the positions of methylated cytosine (mC) of healthy DNA.
Analysis of the genome, transcriptome, proteome and/or methylome is in many respects potentially superior to indirect, conventional methods, e.g. the detection of metabolic products, especially for diagnostic purposes.
In the last twenty years, biological science has developed a comprehensive range of molecular-biological tools for this. In future, therefore, we can expect even wider application of molecular-biological analyses, e.g. in medical and clinical diagnostics, in forensics, in pharmacy in the development and evaluation of medicinal products, in food analysis and in the monitoring of food production, in agriculture in the breeding of useful plants and animals, and in environmental analysis and in many areas of research.
By analyzing the transcriptome, especially the mRNA in cells, the activities of genes can be determined directly. The quantitative analysis of transcript profiles (mRNA profiles) in cells by techniques of modern molecular biology, e.g. real-time reverse-transcriptase PCR (“real-time RT PCR”) or gene expression chip analyses makes it possible for example to detect incorrectly expressed genes, so that e.g. metabolic disorders, infections or any predisposition to cancer can be diagnosed.
Analysis of the genome by molecular biomethods, e.g. PCR, RFLP, AFLP or sequencing makes it possible for example to detect genetic defects or to determine the HLA type and other genetic markers. The analysis of genomic DNA and RNA is also employed for the direct detection of infectious pathogens, such as viruses, bacteria etc. Analysis of the methylome provides indications concerning the activity of particular genes; for example, certain methylation profiles allow conclusions to be drawn regarding predisposition to particular diseases.
In particular, the combination of molecular biomethods with morphological methods is very promising. For example, a tissue sample, for which a particular tumor type has been diagnosed morphologically, can be investigated further by characterization of the genome, transcriptome, proteome and/or methylome, in order to determine a tumor subtype, which in its turn makes it possible to initiate targeted therapy.
An essential precondition for the aforementioned investigations is immediate stabilization of the biological sample after removal from its natural environment, i.e. the conservation of its genomic, transcriptomic, methylomic, proteomic and morphological properties that existed at the time of sample collection.
This applies to the hereditary material DNA, and especially to the less stable RNA, which after collection of the biological sample can be broken down very rapidly by the ubiquitous RNAses. The same applies to DNA methylation profiles, which can be lost or can be falsified by environmental effects after sampling.
Furthermore, after collection of a biological sample, e.g. a section, a biopsy or the like, induction of stress genes and the like can also lead to the synthesis of new mRNA molecules, so that the transcription profile of the cells may be altered.
Stabilization of nucleic acids is necessary in particular in the medical field, because in this case nucleic acid-containing samples are often collected, which can only be investigated further after prolonged storage and transport to a laboratory. In the meantime the nucleic acids contained in the samples may change or even decompose completely. This has a massive influence on the result of subsequent tests or makes them completely impossible. Similarly unfavorable conditions occur e.g. in forensics or in sample collection under field conditions.
Stabilization of proteins is also absolutely essential for investigation of the proteome, as proteins are altered very quickly by modification, e.g. phosphorylation and dephosphorylation, and just like nucleic acids, can be degraded specifically or nonspecifically, or neosynthesis may occur after induction.
For the stabilization of nucleic acids and proteins in compact tissue samples there is yet another difficulty, compared with other biological samples. Tissues are compact, multilayered and heterogeneous with respect to composition, contents and structure. For the stabilization of nucleic acids in tissue samples, the stabilizing reagent must act not only on the surface of cells or within a cell layer, but also deep within the compact, multilayered sample material. In addition, it must be possible to address tissue types that are very varied with respect to their contents and morphology. Differences occur for example in cell structure, membrane structure, boundaries/compartmentations and contents, in particular the protein, carbohydrate or fat content.
Stabilization should then take place without the biological sample being destroyed or having to be destroyed for stabilization. Tissue samples in particular, but also cellular samples are often used for morphological investigation in addition to molecular analysis. This should still be possible after stabilization of the sample. Ideally a substance used for stabilization of the genome, transcriptome, proteome and/or methylome also contributes to the stabilization and preservation of morphological-histological integrity.
It must be possible to provide stabilization by very simple and quick handling of the sample, because on the one hand any pretreatment of the sample that is required (e.g. washing or homogenization) prevents the immediate stabilization of the gene expression profile, since during the delay caused by the pretreatment there may for example be degradation or neosynthesis of RNA. On the other hand, any pretreatment and every additional processing step make it more difficult to use the stabilizing agent. Use anywhere that biological samples can be obtained, e.g. in the operating theater, investigations in the field, in a factory producing foodstuffs, at a crime scene, and the like, is only conceivable if the necessary handling is very simple, without requiring equipment and further sample preparation.